The role of the CPNKEKEC sequence in the β₂ subunit I domain in regulation of integrin α_Lβ₂ (LFA-1)

The α_L I (inserted or interactive) domain of integrin α_Lβ₂ undergoes conformational changes upon activation. Recent studies show that the isolated, activated α_L I domain is sufficient for strong ligand binding, suggesting the β₂ subunit to be only indirectly involved. It has been unclear whether the activity of the α_L I domain is regulated by the β₂ subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the β₂ I domain with a corresponding β₃ sequence, or mutating Lys¹⁷⁴ to Thr, constitutively activates α_Lβ₂ binding to ICAM-1. These mutants do not require Mn²⁺ for ICAM-1 binding and are insensitive to the inhibitory effect of Ca²⁺. We have also localized a component of the mAb 24 epitope (a reporter of β₂ integrin activation) in the CPNKEKEC sequence. Glu¹⁷³ and Glu¹⁷⁵ of the β₂ I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon β₂ integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the α_L I domain did not eliminate the mAb 24 epitope. This confirms that the α_L I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the β₂ subunit I domain. These results suggest that the CPNKEKEC sequence of the β₂ I domain is involved in regulating the α_L I domain.